Comparative Tn-Seq reveals common daptomycin resistance determinants in Staphylococcus aureus despite strain-dependent differences in essentiality of shared cell envelope genes

Antibiotic-resistant Staphylococcus aureus remains a leading cause of antibiotic resistance-associated mortality in the United States. Given the reality of multi-drug resistant infections, it is imperative that we establish and maintain a pipeline of new compounds to replace or supplement our current antibiotics. A first step towards this goal is to prioritize targets by identifying the genes most consistently required for survival across the S. aureus phylogeny. Here we report the first direct comparison of gene essentiality across multiple strains of S. aureus via transposon sequencing. We show that mutant fitness varies by strain in key pathways, underscoring the importance of using more than one strain to differentiate between core and strain-dependent essential genes. Despite baseline differences in gene importance, several pathways, including the lipoteichoic acid pathway, become consistently essential under daptomycin exposure, suggesting core vulnerabilities that can be exploited to resensitize daptomycin-nonsusceptible isolates. We also demonstrate the merit of using transposons with outward-facing promoters capable of overexpressing nearby genes for identifying clinically-relevant gain-of-function resistance mechanisms. Together, the daptomycin vulnerabilities and resistance mechanisms support a mode of action with wide-ranging effects on the cell envelope and cell division. This work adds to a growing body of literature demonstrating the nuanced insights gained by comparing Tn-Seq results across multiple bacterial strains. Author summary Antibiotic-resistant Staphylococcus aureus kills thousands of people every year in the United States alone. To stay ahead of the looming threat of multidrug-resistant infections, we must continue to develop new antibiotics and find ways of making our current repertoire of antibiotics more effective, including by finding pairs of compounds that perform best when administered together. In the age of next-generation sequencing, we can now use transposon sequencing to find potential targets for new antibiotics on a genome-wide scale, identified as either essential genes or genes that become essential in the presence of an antibiotic. In this work, we created a compendium of genes that are essential across a range of S. aureus strains, as well as those that are essential in the presence of the antibiotic daptomycin. The results will be a resource for researchers working to develop the next generation of antibiotic therapies.


Introduction
important in cell division, autolysin regulation, and virulence, among other processes 217 [31]. The LTA pathway consists of five genes: pgcA, gtaB, ugtP, ltaA, and ltaS ( Fig 2B). 218 The first three genes encode proteins involved in the biosynthesis of diglucosyl-219 diacylglycerol (Glc2DAG), the membrane anchor on which LTAs are assembled.  We tested individual deletion mutants in the LTA pathway to confirm the Tn-Seq 236 results. We were able to delete ugtP from USA300-TCH1516, consistent with results in 237 other USA300 strain backgrounds, but a spot dilution assay showed that the mutant 238 was less fit than the corresponding mutant in MW2 (Fig 3B). We also found that ltaS is dispensable in MW2, but essential in other strains ( Fig 4C). Although the MW2ΔltaS 240 strain was more temperature-sensitive than MW2 wildtype, it grew on plates even at 241 37°C (S3 Fig). Because lethality due to ltaS deletion can be suppressed by deletion of 242 gdpP, clpX, and sgtB [37][38][39], we sequenced the transposon library parent strain but 243 found no nonsynonymous SNPs in sgtB or gdpP and only one nonsynonymous SNP in 244 clpX. The amino acid difference in ClpX is unlikely to explain the observed phenotype, 245 as it is located in an unstructured region of the protein and the amino acid is not strictly 246 conserved in the S. aureus phylogeny. Moreover, we did not find any evidence for a 247 duplication of ltaS in MW2 and confirmed that the MW2ΔltaS mutant does not produce 248 LTAs ( Fig 3D). The explanation for why LtaS is nonessential in MW2 must lie elsewhere 249 in the genome and will need to be investigated further. While antibiotics targeting essential genes are invaluable, it may also be 254 advantageous to have compounds that resensitize bacteria to existing antibiotics, 255 though they may not be antimicrobial in vitro on their own. To find targets for such division regulators, gpsB, ezrA, and noc . A number of these 286 depleted genes have been previously associated with daptomycin susceptibility, 287 including mprF [60,61], graRS/vraFG [49,62], and ezrA [63], but some that were 288 substantially depleted in all five strains have not been connected to daptomycin, 289 including gpsB and ltaA. We confirmed that deletion of ltaA sensitizes HG003, MW2, 290 and USA300-TCH1516 to daptomycin and that the sensitivity can be reversed by ltaA 291 complementation ( Fig 5B, S4 Fig). Therefore, Glc2DAG-LTA in the cell membrane 292 contributes to S. aureus survival in the presence of daptomycin, and it may be possible 293 to exploit this observation to overcome daptomycin nonsusceptibility or limit its 294 development.

295
Just as there were more shared than unique depleted genes for each strain, 296 there were more shared than unique upregulated genes ( Fig 4A). The outward-facing 297 promoters in several of our transposon constructs can upregulate downstream genes, 298 allowing us to identify genes that confer a fitness advantage when upregulated. They 299 are detected by strand-biased read enrichments in promoter-containing constructs, and 300 we have previously confirmed that these strand-biased read enrichments, or 301 upregulation signatures, do reflect increased expression of proximal genes [19]. Often 302 the genes identified this way are the targets of the antibiotic itself or reveal gain-of-303 function resistance mechanisms. Standard transposon libraries cannot identify gain-of-304 function resistance mechanisms, although loss-of-function resistance mechanisms can 305 be identified through intragenic read enrichments. Here we found that the genes 306 enriched in reads upon daptomycin exposure belonged to a wide assortment of cellular 307 processes and we could not discern a pattern amongst them (S6 Table). independent mutations that result in changes to the cell envelope [47], and many of the 312 mutations are thought to be gain-of-function mutations that result in increased activity.  Individual mutants need to be tested to confirm that the proximal gene is most 330 responsible for the phenotype. For example, we identified an upregulation signature for ugtP in USA300-TCH1516 and in MSSA476. The ugtP gene is upstream of ltaA in an 332 operon, so it is unclear whether upregulating ugtP on its own would recapitulate the 333 transposon-induced fitness advantage or whether ltaA upregulation is also necessary.

334
Regardless of whether upregulation of one or both genes in the operon drives the 335 fitness advantage, the result is consistent with findings from the depletion analysis that 336 Glc2DAG-LTA is important for withstanding daptomycin stress.  pneumoniae, and Pseudomonas aeruginosa [18,26,72].

364
In the present study, we reveal how complicated the concept of gene essentiality 365 can be. We found that the LTA pathway, which has always been presumed to be 366 essential for all S. aureus strains, was variably essential under normal growth 367 conditions, ranging from required in USA300-TCH1516 to expendable in MW2. We 368 suspect that intracellular Glc2DAG has an unidentified function that USA300-TCH1516 depends upon for survival because the Glc2DAG biosynthesis genes (pgcA, gtaB, and 370 ugtP) contained almost no transposon reads, whereas ltaA, which encodes the flippase 371 responsible for translocating Glc2DAG to the cell surface, contained many insertions.  if there were at least 100 reads in the control file, the q-value was less than 0.05, and 525 the treated:control read ratio was less than 0.1. An enriched gene was considered to be 526 a hit if there were at least 100 normalized reads in the treated file, the q-value was less 527 than 0.05, and the read ratio was greater than ten. We then selected one representative 528 daptomycin concentration for each strain in each medium, chosen to have a similar selective pressure (Table 2). Specifically, the highest concentration file with twenty-five 530 or fewer 10-fold depleted genes with a significant q-value was chosen.  actual value for that gene, and a q-value was obtained using the Benjamini-Hochberg 547 method. We defined an upregulation signature as any UR in which the q-value for the 548 DNA strand matching the gene direction was less than 0.05, there was more than one 549 TA site in the UR, and the opposing strand had a read difference less than the 90 th 550 percentile for all URs. Again, only those samples shown in Table 2 were included.

580
Genomic data was then analyzed to find SNPs, insertions, and deletions.

581
MetaPhlAn2 was used to verify that the DNA was not contaminated. Sequences were 582 then aligned to the appropriate NCBI reference genome using the Burrow-Wheels  To construct an Atet-inducible ltaA construct, the ltaA gene (SAOUHSC_00952) 609 and the ugtP-ltaA operon ribosome binding site were amplified from HG003 genomic 610 DNA using primers ltaA-F and ugtP-R and cloned into pTP63 to generate pTP63-ltaA.

611
As described above, the pTP63-ltaA was transduced into wildtype HG003 and MW2, and the resulting strains were then transduced with a ΔltaA construct marked with a 613 kanamycin resistance gene to generate the inducible ltaA strains [87].

614
To construct an Atet-inducible ltaS construct, the ltaS gene and its ribosome 615 binding site were PCR amplified using the primers ltaS-F and ltaS-R and cloned into 616 pTP63 to make pTP63-ltaS. To make an inducible ltaS strain, pTP63-ltaS and then a 617 ΔltaS construct marked with an erythromycin resistance gene were transduced into 618 wildtype HG003, MW2, and USA300-TCH1516 as described above [37]. The ltaS 619 mutants were confirmed by PCR using the primers ltaS-CA and ltaS-CB.   found to be essential for all three, two, or one of the MRSA strains (vertical bars, with 994 black dots denoting strains for which the genes are essential). Only MRSA strains were included for simplicity, but a similar plot with the MSSA strains is included in the 996 supporting information. Note that gene essentiality is functionally defined as a lack of